antioxidants
Article
Pro-Angiogenic Effects of Natural Antioxidants Extracted from
Mango Leaf, Olive Leaf and Red Grape Pomace over
Endothelial Colony-Forming Cells
Ismael Sánchez-Gomar 1,2,† , Josefa Benítez-Camacho 1,2,† , Cristina Cejudo-Bastante 3,4 , Lourdes Casas 3, * ,
Rafael Moreno-Luna 4 , Casimiro Mantell 3 and Mª Carmen Durán-Ruiz 1,2, *
Citation: Sánchez-Gomar, I.;
Benítez-Camacho, J.;
Cejudo-Bastante, C.; Casas, L.;
Moreno-Luna, R.; Mantell, C.;
Durán-Ruiz, M.C. Pro-Angiogenic
Effects of Natural Antioxidants
Extracted from Mango Leaf, Olive
Leaf and Red Grape Pomace over
Endothelial Colony-Forming Cells.
Antioxidants 2022, 11, 851. https://
doi.org/10.3390/antiox11050851
Academic Editor: Andrei Mocan
Received: 13 April 2022
Accepted: 24 April 2022
Published: 27 April 2022
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Attribution (CC BY) license (https://
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4.0/).
Antioxidants 2022, 11, 851. https://doi.org/10.3390/antiox11050851
1 Biomedicine, Biotechnology and Public Health Department, University of Cadiz, 11002 Cadiz, Spain;
ismael.sanchez@gm.uca.es (I.S.-G.); josefa.benitezcamacho@alum.uca.es (J.B.-C.)
2 Institute of Research and Innovation in Biomedical Sciences of Cadiz (INIBICA), 11009 Cadiz, Spain
3 Chemical Engineering and Food Technology Department, Science Faculty, Wine and Agrifood Research
Institute (IVAGRO), University of Cadiz, 11519 Cadiz, Spain; cristina.cejudo@gm.uca.es (C.C.-B.);
casimiro.mantell@uca.es (C.M.)
4 Laboratory of Neuroinflammation, National Paraplegics Hospital, SESCAM, 45071 Toledo, Spain;
rmluna@sescam.jccm.es
* Correspondence: lourdes.casas@uca.es (L.C.); maricarmen.duran@gm.uca.es (M.C.D.-R.);
Tel.: +34-956-012-727 (M.C.D.-R.)
† These authors contributed equally to this work.
Abstract: Cardiovascular diseases remain the leading cause of death worldwide, mainly triggered by
the formation of atherosclerotic plaques that reduce blood flow. Angiogenic cell therapy based on
endothelial colony forming cells (ECFCs) constitutes a promising alternative to promote vascular
revascularization; however, under the oxidative environment that prevails in ischemic areas, these
cells become impaired. Thus, it is necessary to investigate strategies to enhance their regenerative
properties. Antioxidant substances, such as polyphenols, have been shown to be useful for this
purpose. In the current study we evaluated the potential of mango leaves, olive leaves and red
grape pomace extracts, rich in polyphenols, to promote ECFC reparative effects. For this, aqueous
and ethanolic extracts of the aforementioned raw materials were obtained by pressurized liquid
extraction (PLE). After evaluating the polyphenol content and the antioxidant activity, in vitro assays
were carried out, and we found that ethanolic extracts at low concentrations improved angiogenic
capacities of ECFCs and reduced proliferation, apoptosis, and the inflammatory response of these
cells. Overall, mango leaves ethanolic extract provided the most promising results, but all three
extracts ameliorated the functionality of ECFCs.
Keywords: ECFCs; mango leaves; olive leaves; grape pomace; angiogenesis; proliferation; inflammation;
cell therapy; polyphenols; antioxidants
1. Introduction
Cardiovascular diseases (CVDs) are responsible for a high number of annual deaths
worldwide, with atherosclerosis the main triggering factor [1]. Atherosclerosis is character-
ized by a chronic inflammatory response due to the accumulation of fat in the innermost
layer of the arteries, the intima, causing the appearance of atherosclerotic plaques, limit-
ing blood flow, and promoting ischemic events. Indeed, depending on the location and
severity of the plaque, myocardial infarctions, strokes, or peripheral ischemia may oc-
cur [2]. Cell therapy with endothelial colony-forming cells (ECFCs) provides hope in the
revascularization of ischemic areas in atherosclerotic patients [2,3]; however, these cells
become dysfunctional in the presence of risk factors that predispose a patient to the onset of
ischemic disorders [4]. In this sense, current strategies seeking to enhance their regenerative
https://www.mdpi.com/journal/antioxidants
Antioxidants 2022, 11, 851 2 of 15

potential even under pathological conditions are being designed [4]. For example, the incu-
bation with human platelet lysate boosted ECFCs expansion [5], while preconditioning with
fucoidan [6], erythroid growth factor EPO [7], or nicotine [8] improved ECFCs proliferation.
This proliferation is also stimulated by causing an increase in the intracellular Ca2+ [9].
Similarly, the capacity of some polyphenols to ameliorate therapeutic angiogenesis of
endothelial progenitor cells (EPCs) has also been evaluated. Polyphenols are secondary
plant metabolites known for their strong antioxidative properties. They are abundant in
many healthy dietary patterns and play an important role in the prevention of diseases
associated with oxidative stress and inflammation [10]. In CVDs, polyphenols reduce
endothelial dysfunction by inhibiting low density lipoprotein (LDL) oxidation, reducing
reactive oxygen species (ROS) generation, decreasing the production of proinflamma-
tory cytokines and preventing platelet aggregation and the formation of atherosclerotic
plaques [11,12]. Notably, ELN/41 or Proxison, a novel synthetic flavonoid, was able to
improve ECFCs angiogenic expansion when introduced into explants of ischemic choroids
from P8 C57BL/6J mice [13]. Furthermore, the flavonoids quercetin, kaempferol, and
myricetin seem capable of reducing oxidative stress and apoptosis on EPCs in vitro [14].
Quercetin was reported to enhance the viability and migration of EPCs at a dose-dependent
manner by activating the phosphorylation of protein kinase B or AKT, endothelial nitric
oxide synthase (eNOS) and the protein extracellular signal-regulated kinase (ERK) [15].
Alternatively, it was found that genistein promoted proliferation and migration of ECFCs
in vitro, and the transplantation of ECFCs pre-stimulated with this polyphenol into myocar-
dial ischemic sites promoted neovascularization and improved cardiac function in vivo [16].
The application of high-pressure-based extraction techniques such as pressurized
liquid extraction (PLE) has significantly benefited this field, since phenolic extracts are
currently easier to obtain, minimizing solvent consumption and reducing the cost of
purification steps [17–19]. Indeed, thanks to these approaches, many studies have focused
on evaluating the use of mango leaves, olive leaves and red grape pomace extracts, rich in
polyphenols, in the treatment of CVDs [20–22]. Nevertheless, regarding cell therapy, there
is not much information on the use of these extracts on the angiogenic capacities of ECFCs.
Thus, bearing in mind that antioxidant and anti-inflammatory properties of extracts rich
in polyphenols correlate with an improvement in reparative capacities of endothelial cells
(ECs), in the current study we evaluated whether mango leaves, olive leaves, and red grape
pomace extracts can improve angiogenic capacities of ECFCs, along with their effect on
proliferation, differentiation towards more mature ECs, apoptosis, and inflammation.

2. Materials and Methods

2.1. Raw Material
The raw materials used to obtain the extracts were mango leaves, provided by the
Institute for Mediterranean and Subtropical Horticulture ‘La Mayora’ (IHSM) of the Spanish
National Research Council (CSIC; Malaga, Spain), olive leaves (San José de Lora de Estepa
olivarera Sca Coop, Seville, Spain), and red grape pomace (Luis Pérez wineries, Jerez,
Spain). Mango and olive leaves were collected in 2018 and 2019, respectively. All raw
materials were dried under ambient conditions and stored at room temperature in the
absence of light.

2.2. Chemicals and Reagents

Ultrapure water (Milli-Q) and ethanol (96%) (EtOH) were obtained from Panreac
(Barcelona, Spain). 2,2-Diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu reagent, sodium
carbonate, gallic acid monohydrate, potassium chlorate, sodium acetate, and hydrochloric
acid were supplied by Sigma–Aldrich (Steinheim, Germany).

2.3. Pressurized Liquid Extraction (PLE)

Mango leaves (Mangifera indica L. cv Kent), olive leaves (Olea europaea) and red grape
pomace (Vitis vinifera) extracts were obtained by PLE. Extractions were carried out in high
Antioxidants 2022, 11, 851 3 of 15

pressure extractors (SF1000 and SF2000, Thar Technology), fitted with a thermostatted vessel
with a capacity of 1–2 L, two double piston high pressure pumps (Thar Technology model
P100 for carbon dioxide and P50 for the co-solvent), a preheater, a back pressure regulator
valve (BPR) and a cyclonic separator. In order to compare the yields and antioxidant
capacity of the extracts obtained, two solvents were analyzed in the present work, ultrapure
water (Milli-Q) and ethanol (96%), both provided by Panreac (Barcelona, Spain).
The working conditions were set at a pressure of 200 bar, a temperature of 80 ◦ C and
an extraction time of 12 h in static mode, based on previous studies [17,23]. A sample
quantity between 100 and 500 g was introduced depending on the type of extractor used.

2.4. Extraction Yield and Total Polyphenol Content (TPC)

The concentration and the extraction yield were determined by gravimetry. The total
polyphenol content (TPC) was obtained by a test based on the Folin–Ciocalteu method [24],
analysing equivalent gallic acid concentration (GAEq) in the extracts.

2.5. Antioxidant Activity by DPPH Method

The antioxidant activity of the extracts was determined by the method previously
described [25,26], based on the property of the 2,2-diphenyl-1-picrylhydracil (DPPH) free
radical to absorb at a wavelength of 515 nm, and in the loss of its absorption capacity when
it is reduced by an antioxidant substance [27].

2.6. Selection of Extracts

Based on their antioxidant activity, several concentration ranges were tested in a
viability test, to determine in which concentrations cell viability remained (Supplementary
Figure S1). Thus, two concentrations per extract were selected: the lowest at which an
improvement in the viability of the culture was observed, and the highest at which the
culture was kept viable: Mango-H2 O (11.67 and 186.67 µg/mL); Mango-EtOH (4.92 and
26.25 µg/mL); Olive-H2 O (140 and 560 µg/mL); Olive-EtOH (46.67 and 140 µg/mL);
Grape-H2 O (466.67 and 1866.67 µg/mL); Grape-EtOH (11.67–93.34 µg/mL).
Next, an angiogenesis test was performed, to obtain a first approximation of how these
extracts affect the angiogenic capacity of ECFCs and to select only the optimal concentration
at which these extracts promote angiogenesis to a greater extent (Supplementary Figure S2).
Based on these preliminary results, further experiments were carried out with the
lowest concentrations of the ethanolic extracts: Mango-EtOH (4.92 µg/mL), Olive-EtOH
(46.67 µg/mL), grape-EtOH (11.67 µg/mL).

2.7. ECFCs Culture and Cell Characterization

Endothelial colony forming cells (ECFCs) were isolated from white adipose tissue,
as described [28]. Briefly, ECFCs were cultured in 75 cm2 flasks, treated with 1% gelatin
(G1890, Sigma, Steinheim, Germany) and incubated in 20% FBS/ EBM-2 media (CC-3156,
Lonza, Basel, Switzerland) containing 1% penicillin/streptomycin (P/S) and the necessary
growth factors (VEGF, hFGF-B, hEGF and R3-IGF-1), ascorbic acid and heparin, without
hydrocortisone (CC-4147, Lonza). Cells were grown until confluence (90%) was reached.
Experiments were performed on passages 6–7.
Cell identity was confirmed by testing cloning-forming ability, as described [2], and
also by flow cytometry, analyzing several specific antibodies against CD31, CD14, CD90,
CD34, CD45, CD73, CD133, CD309 and CD146 (Supplementary Figure S3). An isotype IgG1
antibody was used for negative control. The full list of antibodies is shown in Supplemen-
tary Table S1. Fluorescence was measured using CytoFLEX cytometer (Beckam Coulter,
West Sacramento, CA, USA) and CytExpert software. Finally, data were analyzed with
FlowJo v10.4 software.
Antioxidants 2022, 11, 851 4 of 15

2.8. Angiogenesis Assay

The effect of the extracts on the angiogenic capacity of ECFCs was determined by a
tube formation assay test, using a matrigel support, as described [29]. Briefly, 15,000 cells
were seeded in µ-plate angiogenesis 96 well (ibidi, Fitchburg, WI, USA, 89,646) pre-coated
with 10 µL Matrigel (BD Bioscience, San Jose, CA, USA, 256,231) as described [2], and incu-
bated with the ethanolic extracts at the preselected concentrations: mango (4.92 µg/mL),
olive (46.67 µg/mL), grape (11.67 µg/mL), in a total volume of 50 µL/well of EBM-2,
5% FBS, 1% P/S. In addition, a negative control, with ECFCs in basal medium (EBM-2.
5% FBS, 1% P/S) was included, as well as an angiogenesis inhibition control with ECFCs
and 15 mM sulforaphane (S4441-5MG, Sigma) and an angiogenesis activation control,
incubating ECFCs with Fibroblast Growth Factor (FGF) at 35 ng/mL (R&D Systems). All
conditions were evaluated in triplicate. Cells were incubated at 37 ◦ C, 5% CO2 in a humid
chamber. Photographs were taken after 24, 48 and 72 h with the 4X objective (AE2000
Series, Motic). Images were analyzed with the Angiogenesis Analyzer plugin of ImageJ
software, measuring the number of meshes, the number of segments, and the total length.

2.9. Proliferation-Differentiation Assay

ECFCs were seeded (35,000 cells/well) in 24-well plates containing round coverslips
(1.3 cm diameter) pre-treated with 1% gelatin, incubated with 500 µL of basal medium for
24 h at 37 ◦ C, 5% CO2 , and then incubated with the extracts at the preselected concentrations,
in triplicates.
After 48 h, cells were washed with PBS 1x, fixed with 4% paraformaldehyde and
permeabilized with 0.2% triton in 1x PBS (0.2% PBT) for 30 min in agitation. Blocking was
made 2.5% bovine serum albumin (BSA), in 0.2% PBT, prior incubation at 4 ◦ C overnight,
with the rabbit anti-Ki67 primary antibody (PA5-16785; Invitrogen, Waltham, MA, USA,
EEUU) diluted 1:500, and the mouse anti-human vWF antibody (MA5-14029, Thermo
Fisher Scientific, Waltham, MA, USA, EEUU) diluted 1:200 in 2.5% BSA. Next, secondary
antibodies anti-rabbit 488 (A-11008; Thermo Fisher Scientific) (1:1000) and goat anti-mouse
555 (A-21422: Thermo Fisher Scientific, EEUU) (1:500) were used, 1 h in the dark at RT,
followed by incubation with 4’,6-diamidino-2-phenylindole (DAPI) (1:5000 in 2.5% BSA)
for nuclei staining. Coverslips were mounted on slides using mounting solution (S302380-2;
Dako-Agilent Technologies, Santa Clara, CA, USA, EEUU). Photographs were taken for
each replica at 10X, with an Olympus IX81 fluorescence microscope, and images were
subsequently analyzed with ImageJ software. DAPI nuclear labelling allowed total cells to
be counted, Ki67 positive staining identified proliferating cells, and von Willebrand factor
(vWF) positive labelling marked differentiated cells. The proliferation and differentiation
percentage of each experimental condition was determined.

2.10. Apoptosis Assay

Apoptosis was evaluated by flow cytometry using V450 Annexin V (AN-V) (560,506, BD
Biosciences) and propidium iodide (IP) (556,463, BD Bioscences). ECFCs (7 × 104 cells/well)
were seeded in 24-well plates and incubated for 24 h as indicated above, and then treated
with the same preselected concentrations of mango, olive and grape, for 24 and 48 h, at 37 ◦ C
and 5% CO2 . A control without extracts was used, in addition to the apoptosis negative
controls (without AN-V and/or IP). After incubation, cells were trypsinized, washed twice
with PBS and centrifuged. Next, cell pellets were resuspended in 100 µL of 1X AN-V
Binding Buffer (556,454; BD Biosciences), and incubated with 4 µL of AN-V and 4 µL of IP
for 30 min at 4 ◦ C in the dark. Apoptotic cells were analyzed using CytoFLEX cytometer
(Beckam Coulter, USA) and CytExpert software. Finally, data were analyzed with FlowJo
v10.4 software.

2.11. Anti-Inflammatory Assay

ECFCs (7 × 105 cells/well) were seeded in 24-well plates and incubated with the ex-
tracts, as described above, and also with tumor necrosis factor alpha (TNF-α) (0.05 mg/mL).
Antioxidants 2022, 11, 851 5 of 15

A negative control without extracts and TNF-α was used, as well as a positive control with
TNF-α without extracts, in addition to the controls for calibration.
After 4 h of incubation, cells were detached and washed with cytometry buffer (1X PBS,
2.5% FBS, 2 mM EDTA). Then, 4 µL of each antibody was added: APC anti-human CD106
or anti-human VCAM-1 (305,809; Biolegend, San Diego, CA, USA) and PE anti-human
E-selectin (322,606; Biolegend). Incubation was carried out for 20 min at 4 ◦ C in the dark.
Samples were analyzed using CytoFLEX cytometer (Beckam Coulter, USA) and CytExpert
software. Finally, data were analyzed with FlowJo v10.4 software.

2.12. Statistical Analysis

Data representation and analysis was performed using GraphPad Prism 9 software
and IBM SPSS statistics 25. Data were verified for normal distribution using the Shapiro–
Wilk test. For data that followed a normal distribution, the homogeneity of variances was
confirmed by the Levene test; differences were calculated using variance analysis ANOVA
for multivariate analysis, followed by Tukey’s test post-hoc analysis. For non-parametric
data, differences between the groups were calculated with Kruskal–Wallis test and Mann–
Whitney U test as post-hoc analysis. Data were represented by box and whisker diagrams,
including the median, minimum and maximum values, as well as individual data points.
Differences were statistically significant with p-values < 0.05.

3. Results
3.1. Chemical and Functional Characterization of Extracts Obtained by Pressurized Liquid
Extraction (PLE)
The global yield and the total phenolic content (TPC) of the extracts obtained by
PLE (mango leaves, olive leaves, and red grape pomace extracts), as well as their antioxi-
dant capacity, was measured by the DPPH essay [25,26], represented in terms of efficient
concentration (EC50 ) and antioxidant activity index (AAI) [26] respectively.
Results confirmed that, in the case of mango leaves and grape pomace extracts, a
higher yield was obtained in the aqueous extracts than in the ethanolic ones, in contrast
to the olive leaves whose higher yield was seen in the ethanolic extract (Figure 1A). TPC
was similar in aqueous and ethanolic olive leaves extracts; however, a greater TPC was
obtained when using ethanol in both mango leaves and grape pomace extracts (Figure 1B).
Overall, the use of pure ethanol led to a more selective extraction of phenolic compounds
than water [30]. Finally, in general terms, a higher antioxidant activity was obtained in the
ethanolic extracts than in the aqueous ones (Figure 1C,D). Mango leaves ethanolic extracts
presented the highest antioxidant activity, followed by red grape pomace and olive leaves
(Figure 1C,D). Full information regarding the characteristics of the extracts (polyphenolic
and anthocyanin content, antioxidant activity) as well as the extraction techniques can be
found in previous works [17,19,31,32].

3.2. Selection of the Extracts’ Working Concentrations

Preliminary tests (Supplementary Figures S1 and S2) suggested that ethanolic extracts
potentiate the angiogenic properties of ECFCs to a greater extent than aqueous ones, espe-
cially when using the following ethanolic extracts concentrations: mango (4.92 µg/mL),
olive (46.67 µg/mL), grape (11.67 µg/mL). Furthermore, the incubation with higher con-
centrations of these extracts reduced the angiogenic capacity. Therefore, we decided to
apply these as the experimental concentrations in our study.

3.3. Extracts Enhance the Angiogenic Capacity of ECFCs

The incubation of ECFCs with the mango, olive or grape pomace ethanolic extracts did
not suggest any significant differences after 24 h incubation with any of the extracts selected,
compared to control ECFCs (with basal culture media) or even after incubation with FGF,
angiogenic activator (Figure 2). After 48 h, an increase in the number of all parameters
(number of meshes, segments, and segment length) was seen only in ECFCs treated with
 Antioxidants 2022, 11, 851 6 of 15

1, x FOR PEER REVIEW
mango. Finally, a general activation of ECFC angiogenic capacity was seen after 72 h of
incubation with all the extracts, presenting a higher number of meshes, segments, and
longer segments than control ECFCs. Nevertheless, mango was the extract that 6stimulated
of 15
the highest pro-angiogenic affect, even greater than the one exerted by the angiogenic
activator (p-values < 0.001 in ECFCs + Mango vs ECFCs + FGF).

Figure 1. Chemical and functional characterization of the extracts obtained by PLE. (A) Graphical
Figure 1. Chemical and functional characterization of the extracts obtained by PLE. (A) Graphical
representation of global yield of the aqueous and ethanolic extracts of mango leaf, olive leaf, and
representation of global yield of the aqueous and ethanolic extracts of mango leaf, olive leaf, and
red grape pomace expressed as g/100 g dry extract (%). (B) Total phenolic content expressed as
red grape pomace expressed as g/100 g dry extract (%). (B) Total phenolic content expressed as g/100
g/100 g dry extract. (C) Antioxidant activity represented by the efficient concentration (EC50 )
g dry extract. (C) Antioxidant
(µg/mL). (D)activity represented
Antioxidant by the
activity index efficient
(AAI) concentration
(µg DPPH/µg (EC50are
extract). Values ) (μg/mL). (D)
represented as the
Antioxidant activitymean
index (AAI) (μg DPPH/μg
± SD. *** p-value < 0.001. extract). Values are represented as the mean ± SD.
*** p-value < 0.001.

3.2. Selection of the Extracts’ Working Concentrations

Preliminary tests (Supplementary Figures S1 and S2) suggested that ethanolic ex-
tracts potentiate the angiogenic properties of ECFCs to a greater extent than aqueous ones,
especially when using the following ethanolic extracts concentrations: mango (4.92
 treated with mango. Finally, a general activation of ECFC angiogenic capacity was seen
after 72 h of incubation with all the extracts, presenting a higher number of meshes, seg-
ments, and longer segments than control ECFCs. Nevertheless, mango was the extract that
Antioxidants 2022, 11, 851
stimulated the highest pro-angiogenic affect, even greater than the one exerted by 7the
of 15
an-
giogenic activator (p-values < 0.001 in ECFCs + Mango vs ECFCs + FGF).

Figure
Figure 2. Effect
2. Effect of of selected
selected extracts
extracts onon
thethe angiogenic
angiogenic process
process of of ECFCs.
ECFCs. (A)(A) Representative
Representative images
images
of of
thethe reticular
reticular structures
structures formed
formed byby ECFCs
ECFCs after
after 24,24,
48 48
andand
72 72 h incubation
h incubation in in basal
basal medium,
medium, with
with
the inhibitor sulforaphane (15 mM), the activator FGF (35 ng/mL), or with mango
the inhibitor sulforaphane (15 mM), the activator FGF (35 ng/mL), or with mango (4.99 µg/mL), (4.99 μg/mL), olive
(46.67 μg/mL), and grape (16.67 μg/mL) ethanolic extracts. (B) Graphical representation of the
olive (46.67 µg/mL), and grape (16.67 µg/mL) ethanolic extracts. (B) Graphical representation of
changes seen for the number of meshes, number of segments, and total length of the segments ana-
the changes seen for the number of meshes, number of segments, and total length of the segments
lyzed in ECFCs treated with mango leaves, olive leaves, and red grape pomace ethanolic extracts
analyzed in ECFCs treated with mango leaves, olive leaves, and red grape pomace ethanolic extracts
after 24, 48, and 72 h. (C) Temporal evolution of the number of meshes in ECFCs treated with the
selected extracts. * p < 0.05; *** p < 0.001.
 Antioxidants 2022, 11, x FOR PEER REVIEW 8 of 15

Antioxidants 2022, 11, 851 8 of 15

after 24, 48, and 72 h. (C) Temporal evolution of the number of meshes in ECFCs treated with the
selected extracts. * p < 0.05; *** p < 0.001.

3.4. Olive,Grape
3.4. Olive, GrapeandandMango
Mango Extracts
Extracts Decrease
Decrease the Proliferation
the Proliferation of ECFCs
of ECFCs and Promote
and Promote Their Their
Differentiation intoMature
Differentiation into MatureECs
ECs
All three
All threeextracts,
extracts,mango,
mango,olive,
olive,and
and grape,
grape, ledledto to a significant
a significant decrease
decrease in proliferation
in prolifera-
and higher differentiation levels in ECFCs incubated 48 h with any of the
tion and higher differentiation levels in ECFCs incubated 48 h with any of the extracts, extracts, compared
to
compared to basal ECFCs. Overall, a greater reduction in proliferation was related toa agreater
basal ECFCs. Overall, a greater reduction in proliferation was related to
increase in differentiation.
greater increase Among
in differentiation. Among thethe
extracts,
extracts, the
thedecrease
decrease in ECFC
ECFCproliferation
proliferation was
was pronounced
less less pronounced withwith mango,
mango, compared
compared to to olive
olive ororgrape
grapeextracts,
extracts,where
where aa 10%
10% decreased
de-
creased proliferation was seen vs control cells (Figure 3). Thus, olive was the extract
proliferation was seen vs control cells (Figure 3). Thus, olive was the extract that promoted that
promoted
greater greater differentiation
differentiation of ECFCsof atECFCs at the expense
the expense of lowerof lower proliferation,
proliferation, followedfollowed
by grape and
mango. In fact, significant differences were observed when comparing the effect the
by grape and mango. In fact, significant differences were observed when comparing of mango
effectolive,
and of mango
sinceand olive,
olive since olive
promoted promoted
notably notably
higher higher anti-proliferation
anti-proliferation and pro-
and pro-differentiation
differentiation effects than mango.
effects than mango.

Figure 3. Effect of the selected extracts on ECFC proliferation and differentiation. (A) Representative
images of Ki67 and vWF staining of ECFCs treated 48 h with the ethanolic extracts of mango
(4.99 µg/mL) and olive (46.67 µg/mL) leaves, and red grape pomace (11.67 µg/mL). (B) Box and
whisker diagrams of the effect of the selected extracts on the proliferation and differentiation of
ECFCs after 48 h of treatment. * p-value: * p < 0.05; **p < 0.01; *** p < 0.001.
 3.5. Extracts Reduce Apoptosis in ECFCs
The incubation of ECFCs for 24 and 48 h with all three extracts promoted a reduction
in the number of pre-apoptotic cells (AN-V+, IP-), compared to basal controls. This anti-
Antioxidants 2022, 11, 851 apoptotic effect was more noticeable after 48 h of treatment with grape and olive extracts, 9 of 15
while the down-regulation in the number of pre-apoptotic cells was lower in ECFCs
treated with mango (Figure 4).
3.5.Similarly, the three
Extracts Reduce extracts
Apoptosis strongly inhibited the number of late apoptotic cells
in ECFCs
(AN+/IP+) after 24 h of
The incubation treatment.
of ECFCs On
for 24 the48other
and h withhand, afterextracts
all three 48 h, although
promotedthe number of
a reduction
apoptotic
in the number of pre-apoptotic cells (AN-V+, IP-), compared to basal controls. This anti- to
cells was still lower in ECFCs treated with olive, mango, or grape, compared
ECFCs control,
apoptotic effectthe highest
was anti-apoptotic
more noticeable effect
after 48 h ofwas seen with
treatment with the olive
grape andleaf extract
olive (** p-
extracts,
value
while< the
0.01).
down-regulation in the number of pre-apoptotic cells was lower in ECFCs treated
with mango (Figure 4).

Figure 4. Effect of the selected extracts on the apoptosis of ECFCs. (A) Representative dot-plots of
mango leaves, olive leaves, and red grape pomace extracts effect on ECFCs apoptosis after 24 and
48 h of treatment. (B) Graphical representation of the effect of mango leaves (4.99 µg/mL), olive
leaves (46.67 µg/mL), and red grape pomace (11.67 µg/mL) on early and late apoptosis in ECFCs
after 24 and 48 h of treatment. * p-value: * p < 0.05; ** p < 0.01; *** p < 0.001.

Similarly, the three extracts strongly inhibited the number of late apoptotic cells
(AN+/IP+) after 24 h of treatment. On the other hand, after 48 h, although the number
of apoptotic cells was still lower in ECFCs treated with olive, mango, or grape, compared
Antioxidants 2022, 11, x FOR PEER REVIEW
Antioxidants 2022, 11, 851
10 of 15
Figure 4. Effect of the selected extracts on the apoptosis of ECFCs. (A) Representative dot-plots10 of of
15
mango leaves, olive leaves, and red grape pomace extracts effect on ECFCs apoptosis after 24 and
48 h of treatment. (B) Graphical representation of the effect of mango leaves (4.99 μg/mL), olive
leaves (46.67 μg/mL), and red grape pomace (11.67 μg/mL) on early and late apoptosis in ECFCs
to ECFCs
after 24 and control, the highest
48 h of treatment. anti-apoptotic
* p-value: * < 0.05; ** <effect was
0.01; *** seen with the olive leaf extract
< 0.001.
(** p-value < 0.01).
3.6. Extracts Exert an Anti-Inflammatory Effect in ECFCs
3.6. Extracts Exert an Anti-Inflammatory Effect in ECFCs
The three extracts exerted an anti-inflammatory effect in ECFCs, significantly de-
Thethe
creasing three extracts of
expression exerted an anti-inflammatory
both adhesion effect in
molecules compared to ECFCs, significantly
control cells de-
with TNFα
creasing the expression of both adhesion molecules compared to control cells with TNFα
(Figure 5). Among the extracts, mango promoted the highest reduction of E-selectin levels
(Figure 5). Among the extracts, mango promoted the highest reduction of E-selectin levels
compared to control cells and also compared to olive or grape. As for VCAM-1, mango
compared to control cells and also compared to olive or grape. As for VCAM-1, mango
was again the extract that decreased its expression the most, followed by grape and olive.
was again the extract that decreased its expression the most, followed by grape and olive.
Figure
Figure 5.
5. Anti-inflammatory
Anti-inflammatory effect
effect of
of selected
selected extracts.
extracts. (A)
(A) Representative
Representative histograms
histograms ofof the
the effect
effect
of
of treatment with TNFα alone and TNFα with the mango (4.99 µg/mL), olive (46.67 µg/mL), and
treatment with TNFα alone and TNFα with the mango (4.99 μg/mL), olive (46.67 μg/mL), and
grape (16.67 μg/mL) ethanolic extracts on the expression of the adhesion molecules E-selectin and
grape (16.67 µg/mL) ethanolic extracts on the expression of the adhesion molecules E-selectin and
VCAM-1, at 4 h. (B) Box and whisker diagrams of the effect of selected extracts on the expression of
VCAM-1, at 4 h. (B) Box and whisker diagrams of the effect of selected extracts on the expression of
adhesion molecules. MFI: Mean Fluorescence Signal. ### Significant differences with respect to the
control condition (-TNFα). * Significant differences between the conditions. * p < 0.05; *** p < 0.001.
Antioxidants 2022, 11, 851 11 of 15

4. Discussion
Therapeutic angiogenesis represents a novel strategy that allows the reconstitution of
the damaged vascular network in CVD patients. Due to their great angiogenic capabilities,
ECFCs are the main candidates for vascular repair approaches; however, when these cells
are exposed to inflammatory and oxidative environments, their regenerative role is ad-
versely affected. Therefore, different studies have evaluated the potential of pre-stimulating
ECFCs with antioxidant substances prior to cellular therapy in order to enhance their
regenerative properties and reduce the negative effects of such pathological environments,
finding promising results [13,16].
In the current study, we analyzed the effect of three extracts rich in polyphenols,
mango leaves, olive leaves, and red grape pomace extracts, over ECFCs. Ethanolic extracts
were chosen rather than aqueous ones since they presented higher antioxidant capacity,
especially with mango leaves. Moreover, the ethanolic extracts also exerted a greater
pro-angiogenic effect than aqueous, at least when using low concentrations. Previous
studies have demonstrated that the use of antioxidant substances such as polyphenols,
such as those previously determined in the extracts analyzed herein, correlates with an
improvement of their angiogenic abilities [13].
Despite this, we also observed that higher ethanolic concentrations decreased the
angiogenic capacity of ECFCs. The anti-angiogenic effect was especially notable when
140 µg/mL of the ethanolic olive leaves extract was used, in agreement with the re-
sults seen with BALB-c mice with breast tumour implants treated for three weeks with
225 mg/kg/day of olive tree leaf extract (from a mixture of aqueous acetate buffer and
acetonitrile as solvent). Such treatment reduced tumour angiogenesis and stimulated
apoptosis of tumoral cells [33]. Thus, based on these preliminary results, we focused on
evaluating the effect of ethanolic mango leaves, olive leaves and red grape pomace extracts
at concentrations of 4.99, 46.67 and 16.67 µg/mL, respectively, over ECFCs.
According to our results, mango was the extract with the strongest angiogenic poten-
tial, probably related to the major polyphenol content and antioxidant activity. Similarly,
previous studies demonstrated that mango leaves extract promotes ECs migration, favoring
the angiogenic process, although these effects were dependant on the isolated polyphenols
present in the extract. Thus, while mangiferin promoted migration, quercetin inhibited it.
Therefore, the balance between different components might be decisive in the effect exerted
by the extract [34]. Future studies should determine the effect of individual mango extracts
components over ECFCs.
Grape extract, rich in anthocyanins and phenolic acids, also exerted a pro-angiogenic
effect over ECFCs, although to a lesser extent than mango. A study analysing the angiogenic
properties of anthocyanins and fatty acids from blueberry extracts (similar to the grape
extract) reported that anthocyanins inhibited angiogenesis, while the isolated phenolic
acids as well as the combined treatment with anthocyanins and phenolic acids promoted
it [35]. Additionally, resveratrol, a polyphenol contained in red grape skin, was found to
enhance angiogenesis on umbilical cord vein endothelial cells (HUVECs) [36], supporting
the results observed in the present study.
Finally, olive extract also improved the angiogenic process in ECFCs, only 72 h after
treatment. Similarly, previous results revealed that low concentrations (1–5 µM) of 3-
hydroxytyrosol, one component of olive extract, stimulated migration and angiogenesis of
ECs in vitro [37].
Despite the above, most researchers have reported an anti-angiogenic role for these
extracts, mainly in cancer-related studies. For instance, mangiferin seems promising
in the treatment of melanoma due to its anti-angiogenic and antimetastatic effects [38].
Similarly, oleuropein administration decreased angiogenesis and lymphangiogenesis in
HUVECs and lymphatic endothelial cells (LECs), as well as prevented tumor progression
in a murine melanoma model [39]. In addition, grape seed extracts (with polyphenolic
composition similar to red grape pomace extract) exerted an anticancer effect in a murine
model of prostate cancer, due to its anti-proliferative, pro-apoptotic and anti-angiogenic
Antioxidants 2022, 11, 851 12 of 15

activity [40]. Therefore, although the conditions that lead the extracts to exhibit a pro- or
anti-angiogenic behaviour remain unclear, this could be explained by either the cell type,
or by the concentration of the extracts, as shown here.
Regarding proliferation, all three extracts showed anti-proliferative and differentiation-
enhancing effects over ECFCs. These effects were stronger in response to the olive extract,
followed by the pomace and mango. The anti-proliferative activity of the extracts can
be supported by a wide range of studies. For instance, extracts from mango leaves or
from other parts of this plant are capable of exerting a protective effect against different
types of breast cancer, through its cytotoxic and anti-proliferative effect, which cannot
be associated with a single component of the extract, but to the synergistic effect of the
different polyphenols present [30,41]. Furthermore, grape pomace and olive leaf extracts
seem to inhibit the proliferation of colon cancer cells [42] and different human carcinoma
cell lines [43], respectively.
Regarding the increase in differentiation, olive leaves extract also promoted the differ-
entiation of human mesenchymal stem cells (hMSCs) towards ECs, increasing the expres-
sion of vascular growth factor endothelial (VEGF), platelet-derived growth factor receptor
(PDGFR) and the endothelial growth factor receptor (VEGFR-1) [44]. Furthermore, resvera-
trol, a polyphenol present in grape skin, induces the differentiation of vascular progenitor
cells to endothelial cells [45].
The increase in differentiation as well as the consequent decrease in proliferation are
related to the pro-angiogenic capacity of the extracts, given that the differentiation towards
more mature ECs promotes the formation of tubular structures, an essential process in
angiogenesis and vasculogenesis [44], as well as mobilization towards the injured area and
vascular repair [46].
The three extracts also decreased apoptosis in ECFCs, more significantly with olive
extract, followed by pomace and mango. Several studies support this anti-apoptotic effect,
which may be associated with its antioxidant and anti-inflammatory capacities, reflect-
ing a protective role [47–49]. The reduction of the apoptotic process would also support
differentiation outcomes at the expense of a decrease in proliferating cells.
Finally, as already mentioned, the three extracts exhibited an anti-inflammatory ac-
tivity on ECFCs, reducing the expression of endothelial adhesion molecules VCAM-1 and
E-selectin. The anti-inflammatory effect of the analyzed extracts has been previously re-
ported [50–52] and, along with the antioxidant activity, may also be related to a protective
effect on ECFCs.
Of note, vWF up-regulation is often associated with endothelial damage and in-
flammation, platelet aggregation, and adhesion, and it is considered as a biomarker of
atherosclerosis and thrombogenesis, among others [53]. In the current study, however,
considering that the extracts reduced the inflammatory response in ECFCs, the increased
expression of vWF should not be associated with endothelial dysfunction, but with greater
maturation of these cells [53].

5. Conclusions
Overall, our findings suggest that low concentrations of mango leaf, olive leaf and red
grape pomace ethanolic extracts can exert a pro-angiogenic, anti-proliferative, and anti-
apoptotic effect on ECFCs. Moreover, these extracts reduced the inflammatory response of
these cells and promoted ECFCs differentiation into more mature cells. It is worth noting
the great pro-angiogenic power of the mango leaves extract, which was much higher
than that exhibited by the other two extracts studied. These effects are closely related
to its antioxidative properties. Further studies are needed to determine whether the pre-
treatment of ECFCs with these extracts could boost cell therapy and revascularization
in vivo.
Antioxidants 2022, 11, 851 13 of 15

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/antiox11050851/s1. Figure S1: Viability tests; Figure S2: Preliminary
angiogenesis test; Figure S3: Characterization of ECFCs. Table S1: Antibodies used for cytometry
analyses and immunofluorescence.
Author Contributions: Conceptualization, L.C., C.M. and M.C.D.-R.; Formal analysis, I.S.-G. and
J.B.-C.; Funding acquisition, R.M.-L., C.M. and M.C.D.-R.; Investigation, I.S.-G., J.B.-C. and C.C.-B.;
Methodology, I.S.-G., J.B.-C., C.C.-B. and R.M.-L.; Resources, C.M. and M.C.D.-R.; Writing—original
draft, I.S.-G., J.B.-C. and M.C.D.-R.; Writing—review & editing, I.S.-G., J.B.-C., L.C., R.M.-L., C.M. and
M.C.D.-R. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Spanish Ministry of Science and Technology, Project PID2020-
116229RB-I00 and European Regional Development Fund (ERDF); and also by the Institute of Health
Carlos III, ISCIII (PI20-00716, PI18-00427), co-funded by European Regional Development Fund “A
way to make Europe”; and the PAIDI-RETOS (PI20-00932), Junta de Andalucía.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article and Supplementary Material.
Acknowledgments: Thanks to the other members of @Celularuca group, for their help and support.
Conflicts of Interest: The authors declare no conflict of interest.

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